Toxin production by Clostridium botulinum.
The endopeptidase activity assay developed for measurement of purified botulinum
neurotoxin type A (BoNT/A) in clinical therapeutic preparations has been adopted to
provide a specific measure of BoNT/A activity in culture supernatants of proteolytic
C. botulinum type A. Electrophoretic studies and inhibition of BoNT/A activity by
anti-A antibody confirmed the specificity of the assay. The minimum detection limit
was 0.2 MLD50/ml indicating the assay as more sensitive than the standard mouse
bioassay or any other in vitro assay available to date. Whilst the assay did not exhibit
any cross reactions with non-proteolytic (saccharolytic) clostridia, proteolytic C.
botulinum types B and F and C. sporogenes showed some cross reactions.
The endopeptidase assay was used to investigate physiological aspects of BoNT/A
production by proteolytic C. botulinum type A strain NCTC 7272. Growth studies at
15°C, 25°C and 37°C with strain NCTC 7272 demonstrated that the first appearance
of BoNT/A (0.1-1.0 MLD50 ml) occurred during mid-late exponential or early
stationary phase of growth. Extracellular BoNT/A formation was not proportional to
viable count. Slightly more BoNT/A was detected at 25°C than 37° or 15°C. The
results of BoNT/A formation by one of the growth curves at 25°C measured by the
endopeptidase assay and mouse bioassays were very similar confirming the specificity
of the assay. A simple method was developed to lyre the cells so that BoNT/A
formation could be subsequently measured in the endopeptidase assay. The data
obtained following lysis of cells and measurement of intracellular BoNT/A showed
that both intracellular BoNT/A and total BoNT/A formation is not constitutive but are
more closely proportional to viable count than extracellular BoNT/A. Release of
BoNT/A from cells was not associated with autolysis. The conversion of BoNT/A
from the single-chain to dichain form during growth has been measured.
The use of the endopeptidase assay has been also exploited to study BoNT/A
formation by this strain within the population of cells. There was only a four-fold
difference in BoNT/A production by cells of strain NCTC 7272, and further work in
this area is warranted.
Attempts were made to use MAPs for the production of monoclonal antibodies to
SNAP-25 following cleavage by BoNT/E. Whilst the outcome was unsuccessful, the
soundness of the principle was demonstrated